CN104254243A - Novel fungal strain for producing cellulase and saccharification method using same - Google Patents
Novel fungal strain for producing cellulase and saccharification method using same Download PDFInfo
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Abstract
The present invention relates to: the novel fungal strain Pholiota adipose SKU714; a method for producing cellulase from the fungal strain; and a method for the saccharification of cellulose by using the cellulase which is produced. The cellulase which is produced from the fungal strain of the present invention exhibits a far better saccharification yield than do existing saccharifying enzymes and can therefore be put to diverse uses, including in production of bioenergy, the textile industry, the papermaking industry, the detergent industry, the animal feed industry and the food industry.
Description
Technical field
The present invention relates to yellow umbrella (Pholiota adiposa) SKU714 of new strains, for producing the method for cellulase from described bacterial strain, and use the cellulase that produces for the method for diastatic fiber element.
Background technology
Cellulose is organic substance the abundantest on the earth.It is renewable resources, thus without the need to worrying that image-stone is oily or coal is equally exhausted.But cellulose majority is dropped as agriculture and forestry organic waste material, and this is considered to be the main cause of environmental pollution.The annual agriculture and forestry organic waste material worldwide produced more than 3,000,000,000 tons, only just produces more than 800,000,000 tons in Asia.
Because agriculture and forestry organic waste material is primarily of cellulose and hemicellulose composition, if they can be converted into by saccharification the monose comprising glucose, this may have great help for solution food, fuel and environmental problem.
As the commonsense method reclaiming monose from agriculture and forestry organic waste material, knownly add sulfuric acid and carry out the method for saccharification at high temperature under high pressure.But the problem of this method is: the equipment that must use the costliness that can tolerate strong acid and high pressure; Be difficult to because producing various by product be separated and reclaim monose; The process that production cost is high and relevant because needing process by product is that environment is disagreeableness.Because common method for saccharifying is limited for business application, so be studied in the more eco-friendly saccharification of cellulose method that can replace common method for saccharifying.In this respect, developed various carbohydrase and its commercialization has been used for various industrial circle.In addition, just in its application of active research.As carbohydrase, cellulase is widely used in weaving, papermaking, washing agent and feed industry.In addition, its production for low-calorie diet, the fermentation etc. of food waste.
The cell wall of plant is made up of the polymer of such as cellulose (insoluble β-Isosorbide-5-Nitrae-glucan fiber), hemicellulose (polysaccharide of non-cellulose base) and lignin (compound polyphenol polysaccharide).In these compositions, cellulose exists with maximum amount, is secondly that hemicellulose and xylan are as Main Components.Two kinds of compositions account for more than 50% of total phytomass.Cellulose is the homopolymers of the glucose unit connected by β-Isosorbide-5-Nitrae key.For being decomposed into monose, the enzyme of three types is necessary, i.e. inscribe β-Isosorbide-5-Nitrae-dextranase (inscribe β-Isosorbide-5-Nitrae-) [EC 3.2.1.4], circumscribed β-Isosorbide-5-Nitrae-dextranase [EC 3.2.1.91] and β-glucosyl enzym.Endoglucanase is randomly from medial cuts β-Isosorbide-5-Nitrae glucose key, and exoglucanase carries out cutting at non-reducing end glucan is resolved into disaccharides cellobiose.Cellobiose is decomposed into glucose eventually through β-glucosyl enzym.
Cellulase great majority use mould (fungi) to produce.Particularly, aspergillus (Aspergillus) and trichoderma (Trichoderma) is used to carry out suitability for industrialized production cellulase.Although the bacterial strain that trichoderma reesei ZU-02 (Trichoderma reesei ATCC 56764) representatively produces cellulase is in depth studied, its enzyme concentration and activity are all not enough to fully meet industrial requirement.Such as, although from the process of living beings producing and ethanol in next life resource recycling, produce fuel environmental friendliness etc. in there is many advantages, produce ethanol compared with gasoline production costly from lignocellulosic materials.In ethanol production process, the cost producing carbohydrase account for about 60% of total cost of production.
Therefore, strong demand is existed to cellulosic effective saccharifying, particularly to the eco-friendly saccharification of cellulose process using new strains.
Summary of the invention
Technical problem
For the problem solving above-mentioned prior art carries out the present invention.
The new strains providing and show highly active generation cellulase is provided.
The present invention also uses described bacterial strain to produce the method for cellulase for providing.
The present invention is also for providing the method using described cellulase for diastatic fiber element.
Technical scheme
On the one hand, the invention provides yellow umbrella SKU714 (the registration number KCCM 11187P) bacterial strain producing cellulase.
On the other hand, the invention provides the method for generation of cellulase, comprise and cultivate yellow umbrella SKU714 (registration number KCCM 11187P).
According to of the present invention for generation of in the method for cellulase, the medium of the pH 4.5 ~ 5.5 containing corn starch (5 ~ 10g/L), yeast extract (1 ~ 5g/L), potassium dihydrogen phosphate (3 ~ 7g/L), potassium hydrogen phosphate (3 ~ 7g/L), seven water sulfatases (1 ~ 5g/L), thiamine hydrochloride (0.01 ~ 0.03g/L) and carbon source (10 ~ 30g/L) can be used to cultivate.
According to of the present invention for generation of in the method for cellulase, carbon source can be selected from cellulose, cellobiose, straw and microcrystalline cellulose.
According to of the present invention for generation of in the method for cellulase, can cultivate under the condition of the cultivation temperature of the Ventilation Rate of the stir speed (S.S.) of 100-200rpm, 0.8 ~ 1.2vvm and 25 to 30 DEG C.
On the other hand, the invention provides the method using the cellulase produced by yellow umbrella SKU714 bacterial strain to carry out diastatic fiber element.
In the method for diastatic fiber element according to the present invention, saccharification can be carried out under the condition of the temperature of the cellulase concentration of the substrate concentration of 5 ~ 25wt%, 1 ~ 45FPU/g matrix, pH 4 ~ 7 and 50 ~ 80 DEG C.
In the method for diastatic fiber element according to the present invention, white poplar, straw or its mixture can be used as cellulose source or matrix.
Beneficial effect
According to the yellow umbrella SKU714 of new strains of the present invention, it isolates from agaric, produces highly active cellulase.
Because the cellulase produced by new strains according to the present invention demonstrates saccharification yield more better than existing carbohydrase, it is in various applications available, comprises bioenergy production, textile industry, paper industry, detergent industry, feed industry, food industry, the production of low-calorie diet, the fermentation etc. of food waste.
Accompanying drawing explanation
The result that Fig. 1 display is analyzed the genetic affinity between the ITS-5.8S rDNA sequence of bacterial strain of the present invention and similar species.
Fig. 2 a shows along with incubation time is passed, by the yellow umbrella SKU714 bacterial strain of per unit enzyme amount decompose filter paper measure beta-glucosidase activity (-●-), the result of cellobiohydrolase activity (-zero-), endoglucanase activity (-▲-) and glucose yield (-Δ-).
Fig. 2 b shows along with incubation time is passed, measure yellow umbrella SKU714 bacterial strain xylanase activity (-●-), the result of laccase activity (-zero-), Mannanase Activity (-▲-) and lignin peroxidase activity (-Δ-).
The activity that Fig. 3 a shows the β-glucosyl enzym that yellow umbrella SKU714 bacterial strain produces depends on pH.
The activity that Fig. 3 b shows the β-glucosyl enzym produced by yellow umbrella SKU714 bacterial strain depends on temperature.
Embodiment
The present invention is described in more detail by embodiment.But scope of the present invention is not embodiment restriction.
Embodiment 1: screening produces the bacterial strain of cellulase
For screening produces the bacterial strain of cellulase, the agaric culture of 10 μ L is suspended in the physiological saline of 10mL.By the gained suspension (1x10 of 10 μ L
4cfu mL
-1) be laid in the potato dextrose agar of carboxymethyl cellulose containing 2%, and hatch 3 days at 27 DEG C.After solid agar medium forms bacterium colony, plate dyes by the Congo red of use 0.1%, then decolours with 1M sodium chloride.Then, by selecting to have the agaric bacterial strain being screened production of cellulose enzyme by hydrocellulose at the bacterium colony of periphery of bacterial colonies generation haloing.
Screen preliminary bacterial strain (S1-S6) by this method.Use existing production strain Trichoderma reesei ZU-02 in contrast (C), containing after the solid agar medium of carboxymethyl cellulose is tested, from filtering out bacterial strain the S4 bacterial strain selected and show best cellulose degradation ability.
Embodiment 2: qualification bacterial strain
For identifying the S4 bacterial strain filtered out in embodiment 1, by Korean Culture Center, ITS-5.8S rDNA sequence is analyzed.The ITS-5.8S rDNA sequence designations of S4 bacterial strain is SEQ ID NO:1.
As the result analyzing genetic affinity between the ITS-5.8S rDNA sequence of S4 bacterial strain and similar species, described S4 identification of strains is yellow umbrella (Fig. 1).
Described S4 Strain Designation is " yellow umbrella SKU714 (Pholiota adiposa SKU714) " and is deposited in Korean Culture Center on April 20th, 2011 with registration number KCCM 11187P under budapest treaty.
Embodiment 3: the optimization of medium is with production of cellulose enzyme
(1) activity of cellulase depends on carbon source
The activity of testing yellow umbrella SKU714 bacterial strain generation cellulase in 7-L fermentation tank depends on carbon source.Cellulose, glucose, lactose, maltose, cellobiose, carboxymethyl cellulose, sucrose, xylan, rice straw and microcrystalline cellulose are used as carbon source.
After yellow umbrella SKU714 inoculation is in the 50mL flask containing the full culture medium of 50mL (potato starch 4g/L, dextrose 20g/L), bacterial strain is cultivated 5 days at 25 DEG C with 150rpm in shaking table.50mL culture is seeded in the flask of the 50ml containing somatomedin (corn starch be 8g/L, yeast extract 2g/L, potassium dihydrogen phosphate 5g/L, potassium hydrogen phosphate 5g/L, epsom salt 3g/L, thiamine hydrochloride 0.02g/L and carbon source are 20g/L, pH5), cultivates 7 days in 150rpm, 25 DEG C and pH5.
The yellow beta-glucosidase activity of umbrella SKU714 bacterial strain is measured to often kind of carbon source and the result of glucose production is shown in Table 1.
Table 1
As seen from Table 1, excellent cellulase activity is obtained when cellulose, cellobiose, rice straw and microcrystalline cellulose are used as carbon source.Maximum cellulase activity is obtained when cellulose is used as carbon source.
(2) cellulase activity depends on nitrogenous source
The activity of testing yellow umbrella SKU714 bacterial strain generation cellulase in 7-L fermentation tank depends on nitrogenous source.Yeast extract, peptone, corn starch, urea, ammonium sulfate, potassium nitrate, sodium nitrate and tryptone are used as nitrogenous source.
The result of beta-glucosidase activity and glucose production that often kind of carbon source in 5g/L concentration measures yellow umbrella SKU714 bacterial strain is shown in table 2.
Table 2
As seen from Table 2, when yeast extract, corn starch and tryptone are used as nitrogenous source, excellent cellulase activity is obtained.When yeast extract is used as nitrogenous source, obtain maximum cellulase activity.
Embodiment 4: optimization condition of culture is for generation of highly active enzyme
(1) optimization of condition of culture when white poplar is used as matrix
Use medium containing corn starch (8g/L), yeast extract (2g/L), potassium dihydrogen phosphate (5g/L), potassium hydrogen phosphate (5g/L), epsom salt (3g/L), thiamine hydrochloride (0.02g/L) and white poplar (20g/L) in 7L fermentation tank, optimization condition of culture.PH by 3 be changed to 7 and cultivation temperature be changed to 35 DEG C from 20 time compare cellulase activity.Maximum cellulase activity is obtained when pH 5 and 25-30 DEG C.
In addition, under optimized condition of culture, in the medium containing white poplar matrix, in (pH5,25 DEG C), measure the activity (Fig. 2 a and 2b) of often kind of cellulase along with incubation time passing.Fig. 2 a shows, and by passing along with incubation time, measuring and decomposing the β-glucosyl enzym of filter paper, cellobiohydrolase and the activity of endoglucanase and the result of glucose production by per unit enzyme amount.Fig. 2 b shows, and along with incubation time is passed, measures the result of activity change in zytase, laccase, mannase and lignin peroxidase.
(2) cellulase production and active optimization when straw is used as matrix
The medium containing corn starch (8g/L), yeast extract (2g/L), potassium dihydrogen phosphate (5g/L), potassium hydrogen phosphate (5g/L), epsom salt (3g/L), thiamine hydrochloride (0.02g/L) and straw (20g/L) is used to carry out optimization to condition of culture in 7L fermentation tank.PH by 3 change to 7 and cultivation temperature change to 35 DEG C from 20 time compare cellulase production.Maximum cellulase production is obtained when pH 5 and 25 ~ 30 DEG C.
In addition, pH by 3 be changed to 7.5 and temperature change to 85 DEG C from 40 time compare β-Isosorbide-5-Nitrae-glucosidase activity.Result is presented in Fig. 3 a and 3b.From Fig. 3 a and 3b, obtain the activity of maximum cellulase pH 5 and 65 DEG C.
Embodiment 5: the analysis of saccharification yield
Generally speaking, the lignocellulose be included in plant only uses enzymatic hydrolysis can not with high yield by saccharification.For this reason, for increasing by the cellulose hydrolysis efficiency of cellulase, before enzymatic hydrolysis by preprocess method by lignin and hemicellulose fragmentation.In embodiment 5, for pretreatment, 10g straw is joined in the flask of the sodium hydroxide solution of the 2wt% containing 40ml, react 1 hour at 85 DEG C.Then, by the filter of 0.45uM, straw is filtered, 65 DEG C of dryings.
In order to find optimized saccharification condition, test when changing enzyme concentration, substrate concentration, reaction temperature and reaction pH.
First, the cellulase at the pretreated straw of various concentration and various concentration is joined in the sodium-acetate buffer (pH5.0) of the 0.1M of 20mL.React 72 hours with 150rpm in 15-55 DEG C after, reactant mixture is boiled 3 minutes to remove the enzyme of sex change at 100 DEG C, is then cooled to room temperature, under 4000rpm centrifugal 15 minutes.Enzymic activity is measured by reducing sugar method from supernatant.
Reducing in the weight of 105 DEG C of dryings every gram of straw after 24 hours by measuring, determining saccharification yield according to formula 1.
[formula 1]
Saccharification yield (the %)=weight of carbohydrate [in (weight/g matrix of the reducing sugar of generation) the x0.9/ straw] x100
(1) saccharification yield depends on enzyme concentration
When changing enzyme concentration, yellow umbrella SKU714 bacterial strain is used to carry out saccharification when 65 DEG C and pH 6.Result display in table 3.
Table 3
| Enzyme concentration (FPU/g matrix) | Saccharification yield (%) |
| 1 | 9.7 |
| 5 | 23.9 |
| 17.5 | 83.0 |
| 30 | 81.2 |
| 42.5 | 80.6 |
From table 3, obtain best saccharification yield when enzyme concentration is 15-45FPU/g matrix.
(2) saccharification yield depends on substrate concentration
Effect in the white poplar saccharification carried out at the carbohydrase produced by yellow umbrella SKU714 bacterial strain substrate concentration is investigated.Saccharification yield is measured when the initial concentration of white poplar matrix is changed to 27wt% from 1.Result display in table 4.
Table 4
| Substrate concentration (wt%) | Saccharification yield (%) |
| 1 | 43.0 |
| 2 | 63.5 |
| 11 | 81.2 |
| 20 | 83.1 |
| 27 | 51.0 |
From table 4, obtain excellent saccharification yield when the initial concentration of white poplar matrix is 10 ~ 25wt%.Best saccharification yield is obtained when white poplar concentration is 20wt%.
(3) saccharification yield depends on temperature
The effect of temperature in the white poplar saccharification by being undertaken by the carbohydrase of yellow umbrella SKU714 bacterial strain generation is investigated.Saccharification yield is measured at the differential responses temperature of 20,35,50,65 and 80 DEG C.Result display in table 5.
Table 5
| Reaction temperature (DEG C) | Saccharification yield (%) |
| 20 | 33.2 |
| 35 | 45.0 |
| 50 | 75.8 |
| 65 | 82.1 |
| 80 | 71.2 |
From table 5, obtain excellent saccharification yield when saccharification temperature is 50 ~ 80 DEG C.Best saccharification yield is obtained 65 DEG C time.
(4) saccharification yield depends on pH
Effect in the white poplar saccharification carried out at the carbohydrase produced by yellow umbrella SKU714 bacterial strain pH is investigated.Saccharification yield is measured at differential responses pH place 1,3,5,7 and 9.Result is presented in table 6.
Table 6
| pH | Saccharification yield (%) |
| 1 | 20.0 |
| 3 | 62.6 |
| 5 | 81.4 |
| 7 | 84.0 |
| 9 | 39.4 |
From table 6, pH 4 ~ 7 time, obtain excellent saccharification yield.Best saccharification yield is obtained when pH 7.
Embodiment 6: saccharification under optimized condition
(1) the white poplar saccharification using yellow umbrella cellulase to carry out
Use the cellulase produced by yellow umbrella SKU714 bacterial strain under optimal condition, carry out the saccharification of white poplar.Saccharification is carried out under the condition of substrate concentration 10wt%, enzyme concentration 25FPU/g matrix, pH6 and temperature 65 DEG C.With the Novi being derived from trichoderma reesei, the saccharification yield of the cellulase produced by yellow umbrella SKU714 bacterial strain believes that the saccharification yield of cellulase (Celluclast 1.5L) compares in table 7.
Table 7
| Cellulase | Sugar produces (mg/g white poplar) | Saccharification yield (%) |
| Yellow umbrella SKU714 | 672 | 84 |
| Celluclast?1.5L | 242 | 35 |
(2) straw saccharification of yellow umbrella cellulase is used
Use the cellulase produced by yellow umbrella SKU714 bacterial strain under optimized condition, carry out the saccharification of straw.Saccharification 24 hours is carried out under the condition of substrate concentration 10wt%, enzyme concentration 16FPU/g matrix, pH6 and temperature 65 DEG C.The saccharification yield of the saccharification yield that saccharification yield and the Novi being derived from trichoderma reesei of the cellulase produced by yellow umbrella SKU714 bacterial strain believe cellulase (Celluclast 1.5L) and the cellulase that is derived from sliding mushroom (Pholiota nameko) KTCC26163 bacterial strain compares in table 8.
Table 8
| Cellulase | Sugar produces (mg/g straw) | Saccharification yield (%) |
| Yellow umbrella SKU714 | 690 | 88 |
| Trichoderma reesei | 582 | 76 |
| Sliding mushroom | 420 | 56 |
Although sliding mushroom and yellow agaric strain belong to identical Agaricus (Pholiotasp.), they demonstrate diverse saccharification of cellulose effect.But due to low protein production power and low enzymic activity, for being used as carbohydrase, sliding mushroom bacterial strain is then restricted, comprise cellulase, high protein productivity, enzymatic activity high and good heat endurance owing to producing various biomass degrading enzymes, yellow agaric strain according to the present invention is applicable to business application.
[registration number]
Preservation mechanism: Korean Culture Center (abroad)
Registration number: KCCM 11187P
The grade date: 20110420
Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure
International form
Preservation mechanism proves
In accordance with detailed rules and regulations 7.1
Cause: Li Zhengjie
Address: Korea S, Soul 143-701, Huayang hole, Guang Jin district, Chemical Engineering department
BP/4 (KCTC form 17)
Claims (8)
1. produce yellow umbrella (Pholiota adiposa) SKU714 for cellulase, it is with registration number KCCM 11187P preservation.
2., for generation of a method for cellulase, it comprises and produces cellulase by cultivating yellow umbrella according to claim 1 (Pholiota adiposa) SKU714.
3. the method for generation of cellulase according to claim 2, wherein cultivates in the medium of the pH 4.5 ~ 5.5 containing corn starch (5 ~ 10g/L), yeast extract (1 ~ 5g/L), potassium dihydrogen phosphate (3 ~ 7g/L), potassium hydrogen phosphate (3 ~ 7g/L), epsom salt (1 ~ 5g/L), thiamine hydrochloride (0.01 ~ 0.03g/L) and carbon source (10 ~ 30g/L).
4. the method for generation of cellulase according to claim 3, wherein carbon source is selected from cellulose, cellobiose, straw and microcrystalline cellulose.
5. the method for generation of cellulase according to Claims 2 or 3, wherein cultivates under the condition of the cultivation temperature of the stir speed (S.S.) of 100 to 200rpm, the Ventilation Rate of 0.8 ~ 1.2vvm and 25 ~ 30 DEG C.
6., for a method for diastatic fiber element, it comprises and uses the cellulase that produces by cultivating yellow umbrella according to claim 1 (Pholiota adiposa) SKU714 to carry out diastatic fiber element matrix.
7. the method for diastatic fiber element according to claim 6, wherein cellulose matrix is white poplar, straw or its mixture.
8. the method for diastatic fiber element according to claim 6, wherein carries out saccharification under the condition of the temperature of the cellulase concentration of the substrate concentration of 5 ~ 25wt%, 1 ~ 45 FPU/g matrix, pH 4 ~ 7 and 50 ~ 80 DEG C.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20120019335A KR20130097582A (en) | 2012-02-24 | 2012-02-24 | Cellulase producing novel strain and saccharification method using the same |
| KR10-2012-0019335 | 2012-02-24 | ||
| PCT/KR2013/001406 WO2013125886A1 (en) | 2012-02-24 | 2013-02-21 | Novel fungal strain for producing cellulase and saccharification method using same |
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| CN104254243A true CN104254243A (en) | 2014-12-31 |
| CN104254243B CN104254243B (en) | 2017-04-26 |
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| CN201380021450.2A Expired - Fee Related CN104254243B (en) | 2012-02-24 | 2013-02-21 | Novel fungal strain for producing cellulase and saccharification method using same |
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| US (2) | US20150203808A1 (en) |
| EP (1) | EP2829173B1 (en) |
| JP (1) | JP6022606B2 (en) |
| KR (1) | KR20130097582A (en) |
| CN (1) | CN104254243B (en) |
| ES (1) | ES2618730T3 (en) |
| WO (1) | WO2013125886A1 (en) |
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| CN113637594A (en) * | 2021-10-18 | 2021-11-12 | 山东蓬勃生物科技有限公司 | Pholiota adiposa strain YX1, culture method and application thereof |
| CN116334111A (en) * | 2023-03-03 | 2023-06-27 | 上海市农业科学院 | Straw mushroom cellobiose hydrolase and application thereof |
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- 2013-02-21 ES ES13752441.9T patent/ES2618730T3/en active Active
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| CN113637594A (en) * | 2021-10-18 | 2021-11-12 | 山东蓬勃生物科技有限公司 | Pholiota adiposa strain YX1, culture method and application thereof |
| CN113637594B (en) * | 2021-10-18 | 2022-01-07 | 山东蓬勃生物科技有限公司 | Pholiota adiposa strain YX1, culture method and application thereof |
| CN116334111A (en) * | 2023-03-03 | 2023-06-27 | 上海市农业科学院 | Straw mushroom cellobiose hydrolase and application thereof |
| CN116334111B (en) * | 2023-03-03 | 2024-02-09 | 上海市农业科学院 | Straw mushroom cellobiose hydrolase and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6022606B2 (en) | 2016-11-09 |
| EP2829173A1 (en) | 2015-01-28 |
| EP2829173A4 (en) | 2015-10-28 |
| US10196661B2 (en) | 2019-02-05 |
| ES2618730T3 (en) | 2017-06-22 |
| EP2829173B1 (en) | 2016-12-28 |
| KR20130097582A (en) | 2013-09-03 |
| US20150203808A1 (en) | 2015-07-23 |
| WO2013125886A1 (en) | 2013-08-29 |
| JP2015508660A (en) | 2015-03-23 |
| CN104254243B (en) | 2017-04-26 |
| US20170226548A1 (en) | 2017-08-10 |
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